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Nobuko Yoshida - One of the best experts on this subject based on the ideXlab platform.
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Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion
PLoS neglected tropical diseases, 2016Co-Authors: Tatiana M. Clemente, Cristian Cortez, Antônio Da Silva Novaes, Nobuko YoshidaAbstract:Background The question whether metacylic trypomastigote (MT) Forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. Methodology/Principal Findings Metacyclic Forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. Conclusion/Significance Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.
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Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients.
Parasites & vectors, 2016Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Tatiana M. Clemente, Silene Macedo, Nobuko YoshidaAbstract:Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Trypanosoma cruzi Metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with Metacyclic Forms and the number of internalized parasites was counted. All TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI Metacyclic Forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV Metacyclic Forms was higher. Gp90 molecules spontaneously released by TcI Metacyclic Forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate. Our findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of Metacyclic Forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.
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Additional file 1: Figure S1. of Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients
2016Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Tatiana M. Clemente, Silene Macedo, Nobuko YoshidaAbstract:Expression and release of surface gp82 and g90 molecules of T. cruzi Metacyclic trypomastigotes. a Ponceau-S staining of the corresponding Western blot shown in Fig. 1b, to demonstrate equal loading of Metacyclic trypomastigotes samples, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5. The molecular size markers are shown on the left. b Shown in the upper panel is the detergent extract of Metacyclic Forms of the indicated strains, analyzed by Western blot using monoclonal antibodies to gp82 and gp90. Shown in the lower panel is the conditioned medium obtained from strain 515 MT in PBS, pH 7.2, or in citrate buffer, pH 3.5, analyzed by Western blot. c Western blot of detergent extracts of strains 515 and 1434, revealed with mAb 3 F6. Shown in the lower panels is the corresponding Ponceau-S staining d–e Metacyclic Forms of the indicated strains were incubated on ice for 1 h, in absence or in the presence of anti-gp82 mAb 3 F6 (d) or anti-gp90 mAb 1G7 (e). After fixation, the parasites were incubated with Alexa Fluor 488-conjugated anti-IgG and the number of fluorescent parasites was estimated. (TIF 8480 kb)
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Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion
Infection and immunity, 2014Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Mario Augusto Izidoro, Luiz Juliano, Nobuko YoshidaAbstract:Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite Forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage Metacyclic trypomastigotes, the developmental Forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain Metacyclic Forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to Metacyclic Forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the Metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi Metacyclic Forms, through its fibronectin-degrading activity, is implicated in host cell invasion.
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Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.
PloS one, 2013Co-Authors: Tamiris Zanforlin, Nobuko Yoshida, Cristian Cortez, Ethel Bayer-santos, Igor C. Almeida, José Franco Da SilveiraAbstract:Background To invade target cells, Trypanosoma cruzi Metacyclic Forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by Metacyclic Forms. Methods and Findings Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in Metacyclic Forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and Metacyclic Forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in Metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. Conclusions This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during Metacyclic internalization.
Sergio Schenkman - One of the best experts on this subject based on the ideXlab platform.
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expression of trypomastigote trans sialidase in Metacyclic Forms of trypanosoma cruzi increases parasite escape from its parasitophorous vacuole
Cellular Microbiology, 2006Co-Authors: Sergio S C Rubindecelis, Haruki Uemura, Nobuko Yoshida, Sergio SchenkmanAbstract:Summary Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans -sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic Metacyclic trypomastigotes, which correspond to the infective Forms derived from the insect vector. Both Forms have the same capacity to invade mammalian cells, but cell derived trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of Metacyclic Forms. Transfected and non-transfected Metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, Metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected Metacyclics, or Metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, trypomastigotes and Metacyclic Forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.
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Expression of trypomastigote trans‐sialidase in Metacyclic Forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole
Cellular microbiology, 2006Co-Authors: Sergio S. C. Rubin-de-celis, Nobuko Yoshida, Haruki Uemura, Sergio SchenkmanAbstract:Summary Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans -sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic Metacyclic trypomastigotes, which correspond to the infective Forms derived from the insect vector. Both Forms have the same capacity to invade mammalian cells, but cell derived trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of Metacyclic Forms. Transfected and non-transfected Metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, Metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected Metacyclics, or Metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, trypomastigotes and Metacyclic Forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.
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Trans-sialidase from Trypanosoma cruzi epimastigotes is expressed at the stationary phase and is different from the enzyme expressed in trypomastigotes.
Molecular and biochemical parasitology, 1993Co-Authors: Luciana Botelho Chaves, Marcelo R. S. Briones, Sergio SchenkmanAbstract:We have studied the trans-sialidase from insect Forms of Trypanosoma cruzi growing in axenic culture. Log phase epimastigotes expressed little or no trans-sialidase activity, and were unable to incorporate exogenous sialic acid. Transsialidase started to be expressed at the late logarithmic phase, with specific activity increasing steadily as the culture reached the stationary phase. Trans-sialidase was purified from the late log phase epimastigote culture, which contained less than 2% of Metacyclic Forms, yielding a glycoprotein that migrated as a single 90-kDa band in sodium dodecyl sulfate gels. This enzyme features: (1) no reaction with antibodies against the peptide repeats present in the carboxy-terminal of trypomastigote trans-sialidase; (2) positive reaction with antibodies raised against a fragment of trypomastigote trans-sialidase that contains the active site; (3) similar kinetic properties and identical acceptor-donor specificity when compared to the trypomastigote enzyme; and (4) neuraminidase activity in the absence of acceptors. Upon differentiation into Metacyclic Forms, a transsialidase activity containing the carboxy-terminal repeats of the trypomastigote enzyme was released into the medium. These results suggest that epimastigotes express a developmentally regulated trans-sialidase that contains the same catalytic site but lacks the tandem amino acid repeats typical of trypomastigote trans-sialidase.
Cristian Cortez - One of the best experts on this subject based on the ideXlab platform.
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Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion
PLoS neglected tropical diseases, 2016Co-Authors: Tatiana M. Clemente, Cristian Cortez, Antônio Da Silva Novaes, Nobuko YoshidaAbstract:Background The question whether metacylic trypomastigote (MT) Forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. Methodology/Principal Findings Metacyclic Forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. Conclusion/Significance Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.
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Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients.
Parasites & vectors, 2016Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Tatiana M. Clemente, Silene Macedo, Nobuko YoshidaAbstract:Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Trypanosoma cruzi Metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with Metacyclic Forms and the number of internalized parasites was counted. All TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI Metacyclic Forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV Metacyclic Forms was higher. Gp90 molecules spontaneously released by TcI Metacyclic Forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate. Our findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of Metacyclic Forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.
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Additional file 1: Figure S1. of Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients
2016Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Tatiana M. Clemente, Silene Macedo, Nobuko YoshidaAbstract:Expression and release of surface gp82 and g90 molecules of T. cruzi Metacyclic trypomastigotes. a Ponceau-S staining of the corresponding Western blot shown in Fig. 1b, to demonstrate equal loading of Metacyclic trypomastigotes samples, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5. The molecular size markers are shown on the left. b Shown in the upper panel is the detergent extract of Metacyclic Forms of the indicated strains, analyzed by Western blot using monoclonal antibodies to gp82 and gp90. Shown in the lower panel is the conditioned medium obtained from strain 515 MT in PBS, pH 7.2, or in citrate buffer, pH 3.5, analyzed by Western blot. c Western blot of detergent extracts of strains 515 and 1434, revealed with mAb 3 F6. Shown in the lower panels is the corresponding Ponceau-S staining d–e Metacyclic Forms of the indicated strains were incubated on ice for 1 h, in absence or in the presence of anti-gp82 mAb 3 F6 (d) or anti-gp90 mAb 1G7 (e). After fixation, the parasites were incubated with Alexa Fluor 488-conjugated anti-IgG and the number of fluorescent parasites was estimated. (TIF 8480 kb)
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Fibronectin-Degrading Activity of Trypanosoma cruzi Cysteine Proteinase Plays a Role in Host Cell Invasion
Infection and immunity, 2014Co-Authors: Fernando Yukio Maeda, Cristian Cortez, Mario Augusto Izidoro, Luiz Juliano, Nobuko YoshidaAbstract:Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite Forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage Metacyclic trypomastigotes, the developmental Forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain Metacyclic Forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to Metacyclic Forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the Metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi Metacyclic Forms, through its fibronectin-degrading activity, is implicated in host cell invasion.
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Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.
PloS one, 2013Co-Authors: Tamiris Zanforlin, Nobuko Yoshida, Cristian Cortez, Ethel Bayer-santos, Igor C. Almeida, José Franco Da SilveiraAbstract:Background To invade target cells, Trypanosoma cruzi Metacyclic Forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by Metacyclic Forms. Methods and Findings Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in Metacyclic Forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and Metacyclic Forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in Metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. Conclusions This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during Metacyclic internalization.
Ivan Neira - One of the best experts on this subject based on the ideXlab platform.
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Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice
Microbes and infection, 2005Co-Authors: Mauro Cortez, Ivan Neira, Daniele Ferreira, Alejandro O. Luquetti, Anis Rassi, Marcelo R. Silva, Gisela Rodrigues Da Silva Sasso, Nobuko YoshidaAbstract:Abstract Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic Forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, ~threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, Metacyclic Forms of isolate 573, like the CL isolate, produced high parasitemia (> 106 parasites per ml at the peak), killing ~40% of animals, whereas infection with isolate 587 resulted in low parasitemia (
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Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14.
International journal for parasitology, 2004Co-Authors: Vanessa D. Atayde, Ivan Neira, Mauro Cortez, Daniele Ferreira, Edna Freymüller, Nobuko YoshidaAbstract:Abstract We investigated the properties of Metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, Metacyclic Forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate Metacyclic Forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 Metacyclic Forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH 4 Cl, which releases Ca 2+ from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 Metacyclic Forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.
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involvement of trypanosoma cruzi Metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells
Infection and Immunity, 2003Co-Authors: Ivan Neira, Fernando A Silva, Mauro Cortez, Nobuko YoshidaAbstract:Upon oral infection, Trypanosoma cruzi Metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the Metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic Forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the Metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by Metacyclic Forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of Metacyclic Forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of Metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.
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Infection by Trypanosoma cruzi Metacyclic Forms deficient in gp82 but expressing a related surface molecule, gp30.
Infection and immunity, 2003Co-Authors: Mauro Cortez, Ivan Neira, Daniele Ferreira, Alejandro O. Luquetti, Anis Rassi, Vanessa D. Atayde, Nobuko YoshidaAbstract:Trypanosoma cruzi Metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium after oral infection. In this study we analyzed the process of infection by T. cruzi isolates deficient in the expression of gp82, the Metacyclic stage-specific surface glycoprotein implicated in target cell entry in vitro and in promoting mucosal infection in mice after oral challenge. Mice infected by the oral route with Metacyclic Forms of gp82-deficient isolate 569 or 588 developed patent parasitemia but at greatly reduced levels compared to those infected with the gp82-expressing isolate CL. Metacyclic Forms of both isolates expressed gp30, a surface glycoprotein detectable by monoclonal antibody (MAb) 3F6 directed to gp82. Otherwise, the gp82-deficient isolates displayed a surface profile similar to that of the CL isolate and also entered epithelial HeLa cells in a manner inhibitable by MAb 3F6 and dependent on the parasite signal transduction that involved the activation of protein tyrosine kinase and Ca2+ mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca2+ response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of Metacyclic Forms of isolates 569 and 588 by ∼90 and ∼70%, respectively. A cell invasion assay performed in the presence of gastric mucin, mimicking the in vivo infection, showed an inhibition of 70 to 75% in the internalization of gp82-deficient isolates but not of the CL isolate. The recombinant gp82 exhibited an adhesive capacity toward gastric mucin much higher than that of gp30. Taken together, our findings indicate that target cell entry of Metacyclic trypomastigotes can be mediated either by gp82 or gp30 but that efficient mucosal infection depends on the expression of gp82.
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Cell adhesion and Ca2+ signaling activity in stably transfected Trypanosoma cruzi epimastigotes expressing the Metacyclic stage-specific surface molecule gp82.
Infection and immunity, 2003Co-Authors: Patricio M. Manque, Ivan Neira, Vanessa D. Atayde, José Franco Da Silveira, Esteban M. Cordero, Alice T. Ferreira, Marcel I. Ramirez, Nobuko YoshidaAbstract:Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of Metacyclic Forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca(2+) mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca(2+) response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the Metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca(2+) response in HeLa cells, in the same manner as the Metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the Metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in Metacyclic Forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.
Mauro Cortez - One of the best experts on this subject based on the ideXlab platform.
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Expression and cellular localization of molecules of the gp82 family in Trypanosoma cruzi Metacyclic trypomastigotes.
Infection and immunity, 2007Co-Authors: Vanessa D. Atayde, Mauro Cortez, José Franco Da Silveira, Renata T. Souza, Nobuko YoshidaAbstract:A member of the Trypanosoma cruzi gp82 family, expressed on Metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03, the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03 proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of gp82 family members in Metacyclic Forms of CL and G strains, which belong to highly divergent genetic groups. By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to 5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively. Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82 in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization. Anti-C03 antibodies bound predominantly to flagellar components in permeabilized G strain parasites, but in the CL strain the flagellum was not the preferential target for these antibodies. Host cell invasion of Metacyclic Forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or anti-C03 antibodies.
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Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice
Microbes and infection, 2005Co-Authors: Mauro Cortez, Ivan Neira, Daniele Ferreira, Alejandro O. Luquetti, Anis Rassi, Marcelo R. Silva, Gisela Rodrigues Da Silva Sasso, Nobuko YoshidaAbstract:Abstract Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic Forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, ~threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, Metacyclic Forms of isolate 573, like the CL isolate, produced high parasitemia (> 106 parasites per ml at the peak), killing ~40% of animals, whereas infection with isolate 587 resulted in low parasitemia (
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Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14.
International journal for parasitology, 2004Co-Authors: Vanessa D. Atayde, Ivan Neira, Mauro Cortez, Daniele Ferreira, Edna Freymüller, Nobuko YoshidaAbstract:Abstract We investigated the properties of Metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, Metacyclic Forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate Metacyclic Forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 Metacyclic Forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH 4 Cl, which releases Ca 2+ from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 Metacyclic Forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.
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involvement of trypanosoma cruzi Metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells
Infection and Immunity, 2003Co-Authors: Ivan Neira, Fernando A Silva, Mauro Cortez, Nobuko YoshidaAbstract:Upon oral infection, Trypanosoma cruzi Metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the Metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic Forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the Metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by Metacyclic Forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of Metacyclic Forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of Metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.
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Infection by Trypanosoma cruzi Metacyclic Forms deficient in gp82 but expressing a related surface molecule, gp30.
Infection and immunity, 2003Co-Authors: Mauro Cortez, Ivan Neira, Daniele Ferreira, Alejandro O. Luquetti, Anis Rassi, Vanessa D. Atayde, Nobuko YoshidaAbstract:Trypanosoma cruzi Metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium after oral infection. In this study we analyzed the process of infection by T. cruzi isolates deficient in the expression of gp82, the Metacyclic stage-specific surface glycoprotein implicated in target cell entry in vitro and in promoting mucosal infection in mice after oral challenge. Mice infected by the oral route with Metacyclic Forms of gp82-deficient isolate 569 or 588 developed patent parasitemia but at greatly reduced levels compared to those infected with the gp82-expressing isolate CL. Metacyclic Forms of both isolates expressed gp30, a surface glycoprotein detectable by monoclonal antibody (MAb) 3F6 directed to gp82. Otherwise, the gp82-deficient isolates displayed a surface profile similar to that of the CL isolate and also entered epithelial HeLa cells in a manner inhibitable by MAb 3F6 and dependent on the parasite signal transduction that involved the activation of protein tyrosine kinase and Ca2+ mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca2+ response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of Metacyclic Forms of isolates 569 and 588 by ∼90 and ∼70%, respectively. A cell invasion assay performed in the presence of gastric mucin, mimicking the in vivo infection, showed an inhibition of 70 to 75% in the internalization of gp82-deficient isolates but not of the CL isolate. The recombinant gp82 exhibited an adhesive capacity toward gastric mucin much higher than that of gp30. Taken together, our findings indicate that target cell entry of Metacyclic trypomastigotes can be mediated either by gp82 or gp30 but that efficient mucosal infection depends on the expression of gp82.